Contact inhibition of locomotion was discovered by Abercrombie more than 50 years ago and describes the behaviour of fibroblast cells confronting each other in vitro, where they retract their protrusions and change direction on contact1,2. Its failure was suggested to contribute to malignant invasion3,4,5,6. However, the molecular basis of contact inhibition of locomotion and whether it also occurs in vivo are still unknown. Here we show that neural crest cells, a highly migratory and multipotent embryonic cell population, whose behaviour has been likened to malignant invasion6,7,8, demonstrate contact inhibition of locomotion both in vivo and in vitro, and that this accounts for their directional migration. When two migrating neural crest cells meet, they stop, collapse their protrusions and change direction. In contrast, when a neural crest cell meets another cell type, it fails to display contact inhibition of locomotion; instead, it invades the other tissue, in the same manner as metastatic cancer cells3,5,9. We show that inhibition of non-canonical Wnt signalling abolishes both contact inhibition of locomotion and the directionality of neural crest migration. Wnt-signalling members localize at the site of cell contact, leading to activation of RhoA in this region. These results provide the first example of contact inhibition of locomotion in vivo, provide an explanation for coherent directional migration of groups of cells and establish a previously unknown role for non-canonical Wnt signalling.
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We thank M. Tada, M. Tawk, J. Clarke, C.-P. Heisenberg, R. Kelsh, L. Dale and S. Fraser for reagents, constructs and fish lines; C. F. Riaz for scanning electron microscopy images; and M. Bronner-Fraser, M. Raff, J. Green and A. Ridley for comments on the manuscript. This study was supported by grants to R.M. from the Medical Research Council (MRC) and the Biotechnology and Biological Sciences Research Council. H.K.M. and C.C.-F. are MRC and Boehringer Ingelheim Fonds PhD scholarship holders, respectively, and M.M. is an EMBO postdoctoral fellow.
Author Contributions C.C.-F. and R.M. designed the experiments. C.C.-F., H.K.M. and R.M. performed most of the experiments. C.C.-F. and R.M. did the movie analysis. C.C.-F., G.A.D. and R.M. planned and performed the statistical analysis. M.P., S.K., C.C.-F., H.K.M. and R.M. conducted the FRET analysis. M.M. made some of the constructs and the zebrafish transgenic. M.M., C.C.-F., H.K.M. and R.M. performed the PCP localization experiments. C.C.-F., C.S. and R.M. wrote the paper.
Supplementary Movie 1 shows the Transformation of an internal cell into a leading cell. Dissociated NC were re-aggregated to form a large cluster left to migrate. Note that the random migration of the internal cell (blue track) is transformed into directional migration (green track) when there is a free space in front of the cell. 20X; 1 frame/ 2 min.
Supplementary Movie 2 shows the Migration of NC in groups vs individual cells. Dissociated NC cells were cultured as individual cells or re-aggregated in groups and left to migrate. Note the directional migration of the group’s leading cell when is in contact with neighbour cells and the low persistence of the individual cell. 20X; 1 frame/ 2 min.
Supplementary Movie 3 shows NC cells invading mesodermal explants but not other neural crest explants. Confrontational explants of NC/NC or NC/mesoderm were cultured in vitro for 4 hrs. Note that there is no overlap in the NC/NC explants, but the NC completely covers the mesoderm explant. 10X, 0.2frame/min.
Supplementary Movie 4 shows Contact Inhibition of Locomotion of NC cells in vitro. Confronting explants of NC cultured in vitro. Note that once a NC cell touchs another cell from the opposite group it collapses its lamellipodium and changes the direction of its migration. DIC images; 40X; 2 frame/min.
Supplementary Movie 5 shows Contact Inhibition of Locomotion of NC cells in vivo. NC migration was analyzed in vivo in a Sox10-GFP transgenic zebrafish embryo by time-lapse microscopy. Only cells that were initially isolated were analyzed. Note how the cell protrusion retracts after contacting the neighbour cell. 63X, 2 frame/min.
Supplementary Movie 6 shows Contact Inhibition of Locomotion of NC cells in vivo. NC migration was analyzed in vivo in a Sox10-GFP transgenic zebrafish embryo by time-lapse microscopy. Note how the cell changes its direction of movement after contacting the neighbouring cells. 20X, 2 frame/min.
Supplementary Movie 7 shows. Contact Inhibition of Locomotion of NC cells in vivo. NC migration was analyzed in vivo in a Sox10-membraneGFP transgenic zebrafish embryo by time-lapse microscopy. Note the dynamic of thin filopdia and how they retract after contacting the neighbour cell. 100X, 1 frame/min.
Supplementary Movie 8 shows Migration in vitro of control and PCP inhibited NC. NC expressing membrane-GFP was cultured in vitro and analysed by time-lapse microscopy. Control and DshDep+ NC. Note how control cells disperse from the cluster; however they crawl PCP inhibited cells one on the top of each other. Control: 20X; DshDep+: 40X, 1 frame/min.
Supplementary Movie 9 shows Migration of individual cells in vitro. NC expressing membrane-GFP/nuclear-RFP (control) membrane-RFP/nuclear-GFP/DshDep+ (DshDep+) were removed from the embryo and dissociated. Time-lapse analysis of individual cells was performed. Note that both cells types are highly and equally motile. 20X; 1 frame/min.
Supplementary Movie 10 shows Contact Inhibition of Locomotion depends on PCP signalling in vivo. NC migration was analyzed in vivo in a Sox10-GFP transgenic zebrafish embryo injected with Prickle MO by time-lapse microscopy. Note that after cell-cell contact cell protrusion are not retracted but cells remain in close contact and their direction of cell movement is not affected. 63X, 2 frame/min.
Supplementary Movie 11 shows Contact Inhibition of Locomotion depends on PCP signalling in vitro. Collision of a control NC cell (green membrane, red nucleus) with a small group of DshDep+ injected cell (red membranes, green nuclei). Note only the control cell exhibits contact inhibition. DIC images; 20X; 1 frame/min.
In Supplementary Movie 12 Dsh is localized in the cell-cell contact. NC expressing Dsh-GFP were cultured in vitro in confrontational explants. Note that Dsh-GFP is localized in the cell-cell-contact and that after cell-cell interaction the cells move away from each other. DIC and fluorescent images are shown. 40X, 1 frame/min.
Supplementary Movie 13 shows Contact Inhibition of Locomotion depends on RhoA activity. NC cells expressing membrane-GFP/nuclear-RFP cultured in vitro in the presence of the Rock inhibitor Y27632. Note how colliding cells remain in close contact after the collision, cell protrusions do not collapse and that the direction of cell movement is not affected. DIC images; 20X; 1 frame/min.